ab138884 Glucose Oxidase Assay Kit (Fluorometric) Instructions for Use For quantifying Glucose oxidase in solution. This product is for research use only and is not intended for diagnostic use.
Table of Contents 1. Introduction 3 2. Protocol Summary 5 3. Kit Contents 6 4. Storage and Handling 6 5. Additional Materials Required 6 6. Assay Protocol 7 7. Data Analysis 13 8. Troubleshooting 14 1
1. Introduction The glucose oxidase is a dimeric protein that catalyzes the oxidation of beta-d-glucose into hydrogen peroxide and D-glucono-1,5- lactone, which is hydrolyzed to gluconic acid. It is widely used for the determination of glucose in body fluids and in removing residual glucose and oxygen from beverages, food and other agricultural products. Furthermore, Glucose oxidase is commonly used in biosensors to detect glucose. ab138884 provides a quick and sensitive method for the measurement of glucose oxidase in solution. It can be performed in a convenient 96-well or 384-well microtiter plate format and readily adapted to automation without a separation step. The kit uses our AbRed Indicator which enables a dual recordable mode. The fluorescent signal can be easily read by either a fluorescence microplate reader at Ex/Em = 540/590 nm or an absorbance microplate reader at 576 nm. With this kit, we have detected as little as 0.05 mu/ml glucose oxidase in a 100 µl reaction volume. 2
Kit Key Features Sensitive: Detect as low as 0.05 mu/ml glucose oxidase in solution. Continuous: Readily adapted to automation without a separation step. Convenient: Formulated to have minimal hands-on time. No wash step needed. Non-Radioactive: No special requirements for waste treatment. 3
2. Protocol Summary Summary for One 96-well Plate Prepare assay reaction mixture Add glucose oxidase standards or test samples Incubate at 37 o C for 10-30 minutes Monitor the fluorescence increase at Ex/Em = 540/590 nm Note: Thaw all the kit components to room temperature before starting the experiment. 4
3. Kit Contents Components Amount Component A: AbRed Indicator Component B: Assay Buffer Component C: Horseradish Peroxidase (HRP) 1 vial 1 bottle (50ml) 1 vial Component D: Glucose Oxidase 1 vial (100 units) Component E: DMSO Component F: Glucose 1 vial (200 µl) 1 vial 4. Storage and Handling Keep at -20 C. Avoid exposure to light. 5. Additional Materials Required 96 or 384-well microplates: Solid black microplates Fluorescence microplate reader 5
6. Assay Protocol Note: This protocol is for one 96 - well plate. A. Preparation of Stock Solutions: 1. Prepare AbRed Indicator stock solution (250X) by adding 100 l of DMSO (Component E) into the vial of AbRed Indicator (Component A). The stock solution should be used promptly. Any remaining solution should be aliquoted and refrozen at -20 o C. Note: The AbRed Indicator is unstable in the presence of thiols such as dithiothreitol (DTT) and 2- mercaptoethanol. The final concentration of DTT or 2- mercaptoethanol in the reaction should be no higher than 10 μm. The AbRed Indicator is also unstable at high ph (> 8.5). Therefore, the reaction should be performed at ph 7 8. The provided assay buffer (ph 7.4) is recommended. Avoid repeated freeze-thaw cycles. 2. Prepare HRP stock solution (50X) by adding 1 ml of Assay Buffer (Component B) into the vial of Horseradish Peroxidase (Component C). 6
Note: The unused 50X HRP stock solution should be divided into single use aliquots and stored at -20 o C. 3. Prepare 100U/ml Glucose Oxidase stock solution by adding 1 ml of Assay Buffer (Component B) into the vial of Glucose Oxidase (Component D). Note: The unused 100 U/ml glucose oxidase stock solution should be divided into single use aliquots and stored at -20 o C. 4. Prepare 10X Glucose stock solution by adding 5 ml of Assay Buffer (Component B) into the vial of Glucose (Component F). Note: The unused 10X glucose stock solution should be stored at -20 o C. 7
B. Preparation of Assay Reaction Mixture: Prepare assay reaction mixture according to the following tables, protected from light. Table 1 Assay reaction mixture for one 96-well plate (2X) Components Volume 250X AbRed Indicator 20 µl 50X HRP Stock Solution 100 µl 10X Glucose Stock Solution 500 µl Assay Buffer (Component B) 4.3 ml Total volume 5 ml Table 2 Layout of glucose oxidase standards and test samples in a solid black 96-well microplate BL BL TS TS.. GOS1 GOS1.... GOS2 GOS2 GOS3 GOS3 GOS4 GOS4 GOS5 GOS5 GOS6 GOS6 GOS7 GOS7 Note: GOS = Glucose Oxidase Standards, BL = Blank Control, TS = Test Samples. 8
Table 3. Reagent composition for each well Glucose Oxidase Standards Serial Dilutions*: 50 μl Blank Control Assay Buffer (Component B): 50 μl Test Sample 50 μl *Note 1: Add the serially diluted glucose oxidase standards from 0.01 to 10 mu/ml into each well from GOS1 to GOS7 in duplicate. Note 2: High concentration of glucose oxidase (e.g., 100 mu/ml, final concentration) may cause reduced fluorescence signal due to the overoxidation of AbRed Indicator (to a non-fluorescent product). 9
C. Run Glucose Oxidase assay: 1. Prepare a glucose oxidase standard by diluting 2 µl of the 100 U/ml glucose oxidase stock solution into 200 µl of Assay Buffer (Component B) to have 1000 mu/ml glucose oxidase standard solution. And then take 10 μl of 1000 mu/ml glucose oxidase standard solution to perform 1:100 and 1:3 serial dilutions to get 10, 3, 1, 0.3, 0.1, 0.03, 0.01 and 0 mu/ml serially diluted glucose oxidase standards (50 μl/well). A non-glucose oxidase buffer is included as blank control. The final glucose oxidase concentrations should be twofold lower (i.e. 0 to 5 mu/ml). 2. Add 50 μl of assay reaction mixture into each well of glucose oxidase standards, blank control, and test samples to make the total glucose oxidase assay volume of 100 µl/well. Note: For a 384-well plate, add 25 μl of sample and 25 μl of assay reaction mixture into each well. 3. Incubate the reaction for 10 to 30 minutes at 37 o C, protected from light. 10
4. Monitor the fluorescence increase by using a fluorescence plate reader at Ex/Em = 530-570/590-600 nm (optimal Ex/Em = 540/590 nm). Note: The contents of the plate can also be transferred to a white clear bottom plate and read by absorbance microplate reader at the wavelength of 576 5 nm. The absorption detection has lower sensitivity compared to the fluorescence reading. 11
7. Data Analysis The fluorescence in blank wells (with the dilution buffer only) is used as a control, and is subtracted from the values for those wells with the glucoase oxidase reaction. Note: The fluorescence background increases with time, thus it is important to subtract the fluorescence intensity value of the blank wells for each data point. Figure 1. Glucose oxidase dose response was measured with ab138884 in a solid black 96-well plate using a microplate reader. As low as 0.05 mu/ ml glucose oxidase was detected with 30 minutes incubation (n=3). 12
8. Troubleshooting Problem Reason Solution Assay not working Unexpected results Assay buffer at wrong temperature Protocol step missed Plate read at incorrect wavelength Unsuitable microtiter plate for assay Measured at wrong wavelength Samples contain impeding substances Unsuitable sample type Sample readings are outside linear range Assay buffer must not be chilled - needs to be at RT Re-read and follow the protocol exactly Ensure you are using appropriate reader and filter settings (refer to datasheet) Fluorescence: Black plates (clear bottoms); Luminescence: White plates; Colorimetry: Clear plates. If critical, datasheet will indicate whether to use flat- or U-shaped wells Use appropriate reader and filter settings described in datasheet Troubleshoot and also consider deproteinizing samples Use recommended samples types as listed on the datasheet Concentrate/ dilute samples to be in linear range 13
Problem Reason Solution Samples with inconsistent readings Lower/ Higher readings in samples and standards Unsuitable sample type Samples prepared in the wrong buffer Samples not deproteinized (if indicated on datasheet) Cell/ tissue samples not sufficiently homogenized Too many freezethaw cycles Samples contain impeding substances Samples are too old or incorrectly stored Not fully thawed kit components Out-of-date kit or incorrectly stored reagents Reagents sitting for extended periods on ice Incorrect incubation time/ temperature Refer to datasheet for details about incompatible samples Use the assay buffer provided (or refer to datasheet for instructions) Use the 10kDa spin column (ab93349) or Deproteinizing sample preparation kit (ab93299) Increase sonication time/ number of strokes with the Dounce homogenizer Aliquot samples to reduce the number of freeze-thaw cycles Troubleshoot and also consider deproteinizing samples Use freshly made samples and store at recommended temperature until use Wait for components to thaw completely and gently mix prior use Always check expiry date and store kit components as recommended on the datasheet Try to prepare a fresh reaction mix prior to each use Refer to datasheet for recommended incubation time and/ or temperature 14
Standard curve is not linear Incorrect amounts used Not fully thawed kit components Pipetting errors when setting up the standard curve Incorrect pipetting when preparing the reaction mix Air bubbles in wells Concentration of standard stock incorrect Errors in standard curve calculations Use of other reagents than those provided with the kit Check pipette is calibrated correctly (always use smallest volume pipette that can pipette entire volume) Wait for components to thaw completely and gently mix prior use Try not to pipette too small volumes Always prepare a master mix Air bubbles will interfere with readings; try to avoid producing air bubbles and always remove bubbles prior to reading plates Recheck datasheet for recommended concentrations of standard stocks Refer to datasheet and re-check the calculations Use fresh components from the same kit For further technical questions please do not hesitate to contact us by email (technical@abcam.com) or phone (select contact us on www.abcam.com for the phone number for your region). 15
16
17
18
UK, EU and ROW Email: technical@abcam.com Tel: +44 (0)1223 696000 www.abcam.com US, Canada and Latin America Email: us.technical@abcam.com Tel: 888-77-ABCAM (22226) www.abcam.com China and Asia Pacific Email: hk.technical@abcam.com Tel: 108008523689 ( 中國聯通 ) www.abcam.cn Japan Email: technical@abcam.co.jp Tel: +81-(0)3-6231-0940 www.abcam.co.jp Copyright 2016 Abcam, All Rights Reserved. The Abcam logo is a registered trademark. All information / detail is correct at time of going to print. 19